Iron-sulfur clusters as inhibitors and catalysts of viral replication.

A virus hijacks host cellular machineries and metabolites in order to reproduce. In response, the innate immune system activates different processes to fight back. Although many aspects of these processes have been well investigated, the key roles played by iron-sulfur [FeS] clusters, which are among the oldest classes of bio-inorganic cofactors, have barely been considered. Here we discuss how several [FeS] cluster-containing proteins activate, support and modulate the innate immune response to restrict viral infections, and how some of these proteins simultaneously support the replication of viruses. We also propose models of function of some proteins in the innate immune response and argue that [FeS] clusters in many of these proteins act as biological 'fuses' to control the response. We hope this overview helps to inspire future research in the emerging field of bio-inorganic virology/immunology and that such studies may reveal new molecular insight into the links between viral infections and diseases like cancer and neurodegeneration.

Mechanistic insights into the three steps of poly(ADP-ribosylation) reversal.

Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.

Ferritin as a Platform for Creating Antiviral Mosaic Nanocages: Prospects for Treating COVID-19.

Infectious diseases are a continues threat to human health and the economy worldwide. The latest example is the global pandemic of COVID-19 caused by SARS-CoV-2. Antibody therapy and vaccines are promising approaches to treat the disease; however, they have bottlenecks: they might have low efficacy or narrow breadth due to the continuous emergence of new strains of the virus or antibodies could cause antibody-dependent enhancement (ADE) of infection. To address these bottlenecks, I propose the use of 24-meric ferritin for the synthesis of mosaic nanocages to deliver a cocktail of antibodies or nanobodies alone or in combination with another therapeutic, like a nucleotide analogue, to mimic the viral entry process and deceive the virus, or to develop mosaic vaccines. I argue that available data showing the effectiveness of ferritin-antibody conjugates in targeting specific cells and ferritin-haemagglutinin nanocages in developing influenza vaccines strongly support my proposals.

SARS-CoV-2 spike glycoprotein-binding proteins expressed by upper respiratory tract bacteria may prevent severe viral infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major global challenge. The virus infects host cells using its spike glycoprotein (S-protein) and has significantly higher infectivity and mortality rates among the aged population. Here, based on bioinformatic analysis, I provide evidence that some members of the upper respiratory tract (URT) commensal bacteria express viral S-protein -binding proteins. Based on this analysis and available data showing a decline in the population of these bacteria in the elderly, I propose that some URT commensal bacteria hamper SARS-CoV-2 infectivity and that a decline in the population of these bacteria contributes to the severity of infection. Further studies should provide a better understanding of the interaction of URT bacteria and SARS-CoV-2, which may lead to new therapeutic approaches.

ddhCTP produced by the radical-SAM activity of RSAD2 (viperin) inhibits the NAD+ -dependent activity of enzymes to modulate metabolism.

Radical S-adenosylmethionine (SAM) domain-containing protein 2 (RSAD2; viperin) is a key enzyme in innate immune responses that is highly expressed in response to viral infection and inflammatory stimuli in many cell types. Recently, it was found that RSAD2 catalyses transformation of cytidine triphosphate (CTP) to its analogue 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). The cellular function of this metabolite is unknown. Here, we analysed the extra- and intracellular metabolite levels in human induced pluripotent stem cell (hiPSC)-derived macrophages using high-resolution LC-MS/MS. The results together with biochemical assays and molecular docking simulations revealed that ddhCTP inhibits the NAD+ -dependent activity of enzymes including that of the housekeeping enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We propose that ddhCTP regulates cellular metabolism in response to inflammatory stimuli such as viral infection, pointing to a broader function of RSAD2 than previously thought.

Mechanism of Diol Dehydration by a Promiscuous Radical-SAM Enzyme Homologue of the Antiviral Enzyme Viperin (RSAD2).

3'-Deoxynucleotides are an important class of drugs because they interfere with the metabolism of nucleotides, and their incorporation into DNA or RNA terminates cell division and viral replication. These compounds are generally produced by multi-step chemical synthesis, and an enzyme with the ability to catalyse the removal of the 3'-deoxy group from different nucleotides has yet to be described. Here, using a combination of HPLC, HRMS and NMR spectroscopy, we demonstrate that a thermostable fungal radical S-adenosylmethionine (SAM) enzyme, with similarity to the vertebrate antiviral enzyme viperin (RSAD2), can catalyse the transformation of CTP, UTP and 5-bromo-UTP to their 3'-deoxy-3',4'-didehydro (ddh) analogues. We show that, unlike the fungal enzyme, human viperin only catalyses the transformation of CTP to ddhCTP. Using electron paramagnetic resonance spectroscopy and molecular docking and dynamics simulations in combination with mutagenesis studies, we provide insight into the origin of the unprecedented substrate promiscuity of the enzyme and the mechanism of dehydration of a nucleotide. Our findings highlight the evolution of substrate specificity in a member of the radical-SAM enzymes. We predict that our work will help in using a new class of the radical-SAM enzymes for the biocatalytic synthesis of 3'-deoxy nucleotide/nucleoside analogues.

A unifying view of the broad-spectrum antiviral activity of RSAD2 (viperin) based on its radical-SAM chemistry.

RSAD2 (cig-5), also known as viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible), is a member of the radical S-adenosylmethionine (SAM) superfamily of enzymes. Since the discovery of this enzyme more than a decade ago, numerous studies have shown that it exhibits antiviral activity against a wide range of viruses. However, there is no clear picture demonstrating the mechanism by which RSAD2 restricts the replication process of different viruses, largely because there is no direct evidence describing its in vivo enzymatic activity. As a result, a multifunctionality model has emerged. According to this model the mechanism by which RSAD2 restricts replication of different viruses varies and in many cases is not dependent on the radical-SAM chemistry of RSAD2. If the radical-SAM activity of RSAD2 is not required for its antiviral function, the question worth asking is: why does the cellular defence mechanism induce the expression of the radical-SAM enzyme RSAD2, which is metabolically expensive due to the requirement for a [4Fe-4S] cluster and usage of SAM? Here, in contrast to the multifunctionality view, I put forward a unifying model. I postulate that the radical-SAM activity of RSAD2 modulates cellular metabolic pathways essential for viral replication and/or cell proliferation and survival. As a result, its catalytic activity restricts the replication of a wide range of viruses via a common cellular function. This view is based on recent discoveries hinting towards possible substrates of RSAD2, re-evaluation of previous studies regarding the antiviral activity of RSAD2, and accumulating evidence suggesting a role of human RSAD2 in the metabolic reprogramming of cells.

The radical-SAM enzyme Viperin catalyzes reductive addition of a 5'-deoxyadenosyl radical to UDP-glucose in vitro.

Viperin, a radical-S-adenosylmethionine (SAM) enzyme conserved from fungi to humans, can restrict replication of many viruses. Neither the molecular mechanism underlying the antiviral activity of Viperin, nor its exact physiological function, is understood: most importantly, no radical-SAM activity has been discovered for Viperin. Here, using electron paramagnetic resonance (EPR) spectroscopy, mass spectrometry, and NMR spectroscopy, we show that uridine diphosphate glucose (UDP-glucose) is a substrate of a fungal Viperin (58% pairwise identity with human Viperin at the amino acid level) in vitro. Structural homology modeling and docking experiments reveal a highly conserved binding pocket in which the position of UDP-glucose is consistent with our experimental data regarding catalytic addition of a 5'-deoxyadenosyl radical and a hydrogen atom to UDP-glucose.

The workings of ferritin: a crossroad of opinions.

Biochemistry of the essential element iron is complicated by radical chemistry associated with Fe(ii) ions and by the extremely low solubility of the Fe(iii) ion in near-neutral water. To mitigate these problems cells from all domains of life synthesize the protein ferritin to take up and oxidize Fe(ii) and to form a soluble storage of Fe(iii) from which iron can be made available for physiology. A long history of studies on ferritin has not yet resulted in a generally accepted mechanism of action of this enzyme. In fact strong disagreement exists between extant ideas on several key steps in the workings of ferritin. The scope of this review is to explain the experimental background of these controversies and to indicate directions towards their possible resolution.

Spectroscopic evidence for the presence of a high-valent Fe(IV) species in the ferroxidase reaction of an archaeal ferritin.

A high-valent Fe(IV) species is proposed to be generated from the decay of a peroxodiferric intermediate in the catalytic cycle at the di-iron cofactor center of dioxygen-activating enzymes such as methane monooxygenase. However, it is believed that this intermediate is not formed in the di-iron substrate site of ferritin, where oxidation of Fe(II) substrate to Fe(III) (the ferroxidase reaction) occurs also via a peroxodiferric intermediate. In opposition to this generally accepted view, here we present evidence for the occurrence of a high-valent Fe(IV) in the ferroxidase reaction of an archaeal ferritin, which is based on trapped intermediates obtained with the freeze-quench technique and combination of spectroscopic characterization. We hypothesize that a Fe(IV) intermediate catalyzes oxidation of excess Fe(II) nearby the ferroxidase center.

Mass spectrometry approach and ELISA reveal the effect of codon optimization on N-linked glycosylation of HIV-1 gp120.

The genes encoding many viral proteins such as HIV-1 envelope glycoprotein gp120 have a tendency for codons that are poorly used by the human genome. Why these codons are frequently present in the HIV genome is not known. The presence of these codons limits expression of HIV-1 gp120 for biochemical studies. The poor codons are replaced by synonymous codons that are frequently present in the highly expressed human genes to overexpress this protein. Whether this codon optimization affects functional properties of gp120 such as its N-linked glycosylation is unknown. We applied a bottom-up mass-spectrometry-based workflow for the direct measurement of deglycosylated and unglycosylated peptides with putative N-linked glycosylation sites, that is, NxS/T motifs. Using this mass-spectrometry approach in combination with ELISA, it is found that codon optimization significantly reduces the frequency with which the dolichol pyrophosphate-linked oligosaccharide is added by the catalytic subunits of oligosaccharide transferase complex to the glycosylation sites. This reduction affects binding of glycan-dependent broadly neutralizing antibodies. These data are essential for biochemical studies of gp120 and successful development of a vaccine against HIV-1. Furthermore, they demonstrate a mass-spectrometry approach for studying the site-specific N-linked glycosylation efficiency of glycoproteins.

The amyloid precursor protein (APP) does not have a ferroxidase site in its E2 domain.

The ubiquitous 24-meric iron-storage protein ferritin and multicopper oxidases such as ceruloplasmin or hephaestin catalyze oxidation of Fe(II) to Fe(III), using molecular oxygen as oxidant. The ferroxidase activity of these proteins is essential for cellular iron homeostasis. It has been reported that the amyloid precursor protein (APP) also has ferroxidase activity. The activity is assigned to a ferroxidase site in the E2 domain of APP. A synthetic 22-residue peptide that carries the putative ferroxidase site of E2 domain (FD1 peptide) has been claimed to encompass the same activity. We previously tested the ferroxidase activity of the synthetic FD1 peptide but we did not observe any activity above the background oxidation of Fe(II) by molecular oxygen. Here we used isothermal titration calorimetry to study Zn(II) and Fe(II) binding to the natural E2 domain of APP, and we employed the transferrin assay and oxygen consumption measurements to test the ferroxidase activity of the E2 domain. We found that this domain neither in the presence nor in the absence of the E1 domain binds Fe(II) and it is not able to catalyze the oxidation of Fe(II). Binding of Cu(II) to the E2 domain did not induce ferroxidase activity contrary to the presence of redox active Cu(II) centers in ceruloplasmin or hephaestin. Thus, we conclude that E2 or E1 domains of APP do not have ferroxidase activity and that the potential involvement of APP as a ferroxidase in the pathology of Alzheimer's disease must be re-evaluated.

Phosphate accelerates displacement of Fe(III) by Fe(II) in the ferroxidase center of Pyrococcus furiosus ferritin.

The iron-storage protein, ferritin, is widely found in all Domains of life. A conserved diiron center in ferritin catalyzes oxidation of Fe(II) and regulates storage of the resultant Fe(III) oxidation product. When this center is filled with Fe(III), in bacterial or archaeal ferritin the presence of phosphate accelerates the rate of Fe(II) oxidation. The molecular mechanism underlying this stimulatory effect of phosphate is unknown. Using site directed mutagenesis of the residues in the diiron center of the archaeal ferritin from Pyrococcus furiosus we show that phosphate facilitates displacement of Fe(III) by Fe(II) from this site. Therefore, the rate of Fe(II) oxidation increases only when the ferroxidase center is filled with Fe(III).

The catalytic center of ferritin regulates iron storage via Fe(II)-Fe(III) displacement.

A conserved iron-binding site, the ferroxidase center, regulates the vital iron storage role of the ubiquitous protein ferritin in iron metabolism. It is commonly thought that two Fe(II) simultaneously bind the ferroxidase center and that the oxidized Fe(III)-O(H)-Fe(III) product spontaneously enters the cavity of ferritin as a unit. In contrast, in some bacterioferritins and in archaeal ferritins a persistent di-iron prosthetic group in this center is believed to mediate catalysis of core formation. Using a combination of binding experiments and isotopically labeled (57)Fe(II), we studied two systems in comparison: the ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus (PfFtn) and the eukaryotic human H ferritin (HuHF). The results do not support either of the two paradigmatic models; instead they suggest a unifying mechanism in which the Fe(III)-O-Fe(III) unit resides in the ferroxidase center until it is sequentially displaced by Fe(II).

Inhibition and stimulation of formation of the ferroxidase center and the iron core in Pyrococcus furiosus ferritin.

Ferritin is a ubiquitous iron-storage protein that has 24 subunits. Each subunit of ferritins that exhibit high Fe(II) oxidation rates has a diiron binding site, the so-called ferroxidase center (FC). The role of the FC appears to be essential for the iron-oxidation catalysis of ferritins. Studies of the iron oxidation by mammalian, bacterial, and archaeal ferritin have indicated different mechanisms are operative for Fe(II) oxidation, and for inhibition of the Fe(II) oxidation by Zn(II). These differences are presumably related to the variations in the amino acid residues of the FC and/or transport channels. We have used a combination of UV-vis spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry to study the inhibiting action of Zn(II) ions on the iron-oxidation process by apoferritin and by ferritin aerobically preloaded with 48 Fe(II) per 24-meric protein, and to study a possible role of phosphate in initial iron mineralization by Pyrococcus furiosus ferritin (PfFtn). Although the empty FC can accommodate two zinc ions, binding of one zinc ion to the FC suffices to essentially abolish iron-oxidation activity. Zn(II) no longer binds to the FC nor does it inhibit iron core formation once the FC is filled with two Fe(III). Phosphate and vanadate facilitate iron oxidation only after formation of a stable FC, whereupon they become an integral part of the core. These results corroborate our previous proposal that the FC in PfFtn is a stable prosthetic group, and they suggest that its formation is essential for iron-oxidation catalysis by the protein.

Catalysis of iron core formation in Pyrococcus furiosus ferritin.

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)-O-Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation.